mutant tdp43 plasmids  (Addgene inc)

Journal: Bio-protocol

Article Title: High-throughput Flow Cytometry Assay to Investigate TDP43 Splicing Function

doi: 10.21769/BioProtoc.3594

Figure Lengend Snippet: A. The fluorescent protein (FP)-based splicing module and BFP-tagged TDP43 variants are expressed from the same plasmid via a bi-directional promoter (pBI). B. Production of a dually-fluorescent GFP-mCherry fusion protein depends on the presence of functional TDP43, which is required to mediate the skipping of CFTR gene-derived exon (labeled E2).

Article Snippet: Cell lines HEK-293T wild-type cells (ATCC, catalog number: CRL-3216) HEK-293T TARDBP knock-out cells (available upon request) Cell culture 1.5 ml microtubes (Eppendorf, catalog number: 022363204) 60 μm scepter tips for cell counting (Milipore-Sigma, catalog number: PHCC60050) 10 cm tissue culture plates for propagating cell lines (Thermo Scientific, catalog number: 12-556-002) 6-well tissue culture plates for splicing assays (Thermo Scientific, catalog number: 14-832-11) 70 μm cell strainer (Corning, Falcon, catalog number: 08-771-2) 5 ml round-bottom polystyrene tubes (Corning, Falcon, catalog number: 14-959-1A) SH800S flow cytometer setup beads (Sony Biotechnology, catalog number: LE-B3001) HyClone DMEM high glucose (GE Healthcare, catalog number: SH30081FS) supplemented with: 10% FBS (Atlanta Biologicals, catalog number: S11150H) 1 mM sodium pyruvate (Gibco, catalog number: 11-360-070) 2 mM L-glutamine (Gemini Biosciences, catalog number: 400106) 1x MEM non-essential amino acids (Gibco, catalog number: 11-140-076) 40 U/ml penicillin and 40 μg/ml streptomycin (Gemini Biosciences, catalog number: 400109) Sterile 1x PBS at pH 7.4 (Sigma-Aldrich, catalog number: P3813-5x10PAK) Trypsin-EDTA (Gemini Biosciences, catalog number: 400150) X-tremeGENE9 transfection reagent (Roche, catalog number: 06365787001) OptiMEM (Gibco, catalog number: 31985-070) Plasmids (available at https://www.addgene.org/Rajat_Rohatgi/ , unless otherwise noted) Splicing reporter only: pHBS1389 IBB-GFP-mCherry3E (Addgene, catalog number: 118803) Positive control: pHBS1503 [IBB-GFP-mCherry3E]-[BFP-TDP43 WT] (Addgene, catalog number: 133327) Negative control: pHBS1501 [IBB-GFP-mCherry3E]-[BFP-TDP43 RRM] (Addgene, catalog number: 133325) Extensive collection of TDP43 CTD mutants in the splicing reporter ( e.g. , Addgene, catalog numbers: 133328-133333) Backbone for subcloning of CTD mutants into splicing reporter: pHBS1224 [IBB-GFP-mCherry3E]-[BFP-TDP43 CTD EcoRV] (available upon request) Compensation probes: mammalian expression vectors with BFP, GFP and mCherry inserts ( e.g. , Addgene, catalog numbers: 54665, 54759 and 54563)

Techniques: Plasmid Preparation, Functional Assay, Derivative Assay, Labeling

Journal: Bio-protocol

Article Title: High-throughput Flow Cytometry Assay to Investigate TDP43 Splicing Function

doi: 10.21769/BioProtoc.3594

Figure Lengend Snippet: A. BSC-A vs. FSC-A density plot showing all detected events and Gate 1 that selects for cells. B. FSC-H vs. FSC-A density plot and Gate 2 to select for single cells. C. FL1-A (BFP) histogram and Gate 3 to select for transfected cells. D. FL3-A (mCherry) vs. FL2-A (GFP) density plots for full-length, wild-type (WT) TDP43 and TDP43 lacking its RNA-binding domains (RRM).

Article Snippet: Cell lines HEK-293T wild-type cells (ATCC, catalog number: CRL-3216) HEK-293T TARDBP knock-out cells (available upon request) Cell culture 1.5 ml microtubes (Eppendorf, catalog number: 022363204) 60 μm scepter tips for cell counting (Milipore-Sigma, catalog number: PHCC60050) 10 cm tissue culture plates for propagating cell lines (Thermo Scientific, catalog number: 12-556-002) 6-well tissue culture plates for splicing assays (Thermo Scientific, catalog number: 14-832-11) 70 μm cell strainer (Corning, Falcon, catalog number: 08-771-2) 5 ml round-bottom polystyrene tubes (Corning, Falcon, catalog number: 14-959-1A) SH800S flow cytometer setup beads (Sony Biotechnology, catalog number: LE-B3001) HyClone DMEM high glucose (GE Healthcare, catalog number: SH30081FS) supplemented with: 10% FBS (Atlanta Biologicals, catalog number: S11150H) 1 mM sodium pyruvate (Gibco, catalog number: 11-360-070) 2 mM L-glutamine (Gemini Biosciences, catalog number: 400106) 1x MEM non-essential amino acids (Gibco, catalog number: 11-140-076) 40 U/ml penicillin and 40 μg/ml streptomycin (Gemini Biosciences, catalog number: 400109) Sterile 1x PBS at pH 7.4 (Sigma-Aldrich, catalog number: P3813-5x10PAK) Trypsin-EDTA (Gemini Biosciences, catalog number: 400150) X-tremeGENE9 transfection reagent (Roche, catalog number: 06365787001) OptiMEM (Gibco, catalog number: 31985-070) Plasmids (available at https://www.addgene.org/Rajat_Rohatgi/ , unless otherwise noted) Splicing reporter only: pHBS1389 IBB-GFP-mCherry3E (Addgene, catalog number: 118803) Positive control: pHBS1503 [IBB-GFP-mCherry3E]-[BFP-TDP43 WT] (Addgene, catalog number: 133327) Negative control: pHBS1501 [IBB-GFP-mCherry3E]-[BFP-TDP43 RRM] (Addgene, catalog number: 133325) Extensive collection of TDP43 CTD mutants in the splicing reporter ( e.g. , Addgene, catalog numbers: 133328-133333) Backbone for subcloning of CTD mutants into splicing reporter: pHBS1224 [IBB-GFP-mCherry3E]-[BFP-TDP43 CTD EcoRV] (available upon request) Compensation probes: mammalian expression vectors with BFP, GFP and mCherry inserts ( e.g. , Addgene, catalog numbers: 54665, 54759 and 54563)

Techniques: Transfection, RNA Binding Assay

Journal: Bio-protocol

Article Title: High-throughput Flow Cytometry Assay to Investigate TDP43 Splicing Function

doi: 10.21769/BioProtoc.3594

Figure Lengend Snippet: A. Outline of the analysis pipeline. A flow cytometer is used to measure and deconvolute the mCherry, GFP and BFP fluorescence signals of individual cells in a population. Using custom scripts, the splicing efficiency is calculated by taking the ratio of the mCherry to GFP signal of a given cell (Figure 1) and compared to the TDP43 level of the same cell (BFP signal). B. Screenshot highlighting where to change the gate boundaries and cut-offs in the ‘CSA_Gating.nb’ script. C. Screenshot indicating where to provide the file path to the fcs raw data in the ‘CSA_Gating.nb’ script. D. Example density plots generated during evaluation of the ‘CSA_Gating.nb’ script to visualize the gates defined for analysis. E. Screenshot illustrating where in the ‘CSA_Plotting.nb’ script to provide the file path to the csv files generated during evaluation of the ‘CSA_Gating.nb’ script. F. Screenshot highlighting where to adjust the plot scales in the ‘CSA_Plotting.nb’ script. G. Example density plot generated by the ‘CSA_Plotting.nb’ script that depicts splicing efficiency vs. TDP43 levels for wild-type (WT) and RRM TDP43 variants (top) and example violin plot highlighting the splicing efficiencies in the defined BFP signal range (bottom). H. Screenshot of the ‘CSA_Plotting.nb’ script indicating where to define the BFP signal range for the violin plots comparing splicing efficiency.

Article Snippet: Cell lines HEK-293T wild-type cells (ATCC, catalog number: CRL-3216) HEK-293T TARDBP knock-out cells (available upon request) Cell culture 1.5 ml microtubes (Eppendorf, catalog number: 022363204) 60 μm scepter tips for cell counting (Milipore-Sigma, catalog number: PHCC60050) 10 cm tissue culture plates for propagating cell lines (Thermo Scientific, catalog number: 12-556-002) 6-well tissue culture plates for splicing assays (Thermo Scientific, catalog number: 14-832-11) 70 μm cell strainer (Corning, Falcon, catalog number: 08-771-2) 5 ml round-bottom polystyrene tubes (Corning, Falcon, catalog number: 14-959-1A) SH800S flow cytometer setup beads (Sony Biotechnology, catalog number: LE-B3001) HyClone DMEM high glucose (GE Healthcare, catalog number: SH30081FS) supplemented with: 10% FBS (Atlanta Biologicals, catalog number: S11150H) 1 mM sodium pyruvate (Gibco, catalog number: 11-360-070) 2 mM L-glutamine (Gemini Biosciences, catalog number: 400106) 1x MEM non-essential amino acids (Gibco, catalog number: 11-140-076) 40 U/ml penicillin and 40 μg/ml streptomycin (Gemini Biosciences, catalog number: 400109) Sterile 1x PBS at pH 7.4 (Sigma-Aldrich, catalog number: P3813-5x10PAK) Trypsin-EDTA (Gemini Biosciences, catalog number: 400150) X-tremeGENE9 transfection reagent (Roche, catalog number: 06365787001) OptiMEM (Gibco, catalog number: 31985-070) Plasmids (available at https://www.addgene.org/Rajat_Rohatgi/ , unless otherwise noted) Splicing reporter only: pHBS1389 IBB-GFP-mCherry3E (Addgene, catalog number: 118803) Positive control: pHBS1503 [IBB-GFP-mCherry3E]-[BFP-TDP43 WT] (Addgene, catalog number: 133327) Negative control: pHBS1501 [IBB-GFP-mCherry3E]-[BFP-TDP43 RRM] (Addgene, catalog number: 133325) Extensive collection of TDP43 CTD mutants in the splicing reporter ( e.g. , Addgene, catalog numbers: 133328-133333) Backbone for subcloning of CTD mutants into splicing reporter: pHBS1224 [IBB-GFP-mCherry3E]-[BFP-TDP43 CTD EcoRV] (available upon request) Compensation probes: mammalian expression vectors with BFP, GFP and mCherry inserts ( e.g. , Addgene, catalog numbers: 54665, 54759 and 54563)

Techniques: Flow Cytometry, Fluorescence, Generated